Project Summary/Abstract Long non-coding RNAs (lncRNAs), a loosely-defined class of RNAs longer than 200 nucleotides, make up a significant fraction of the eukaryotic transcriptome. Thousands of lncRNAs have now been catalogued in numerous species, but relatively few have been functionally characterized, particularly in animal models. Because of the dearth of molecular evidence of lncRNA function and consistently weak signals of lncRNA conservation, the extent of their impact on organismal biology and disease remains unclear; more functional studies in animal models are necessary to fully understand lncRNA biology. The long term objective of this proposal is to investigate lncRNA functions in the key developmental process of oogenesis in a classic animal model: Drosophila melanogaster. The genetics and development of Drosophila oogenesis are well characterized, facilitating investigations of how lncRNAs may impact specific developmental processes and interact with key oogenesis genes. Further, many aspects of oogenesis are shared across all animals, and thus insights into lncRNA biology from Drosophila will be informative for human biology. This proposal has three specific aims. First, the localization of ovary-enriched lncRNAs will be characterized in oogenesis. Analyses of existing ovary RNA-Seq datasets in Drosophila have identified a set of 28 antisense and intergenic lncRNA loci that are enriched in the ovaries and likely conserved in multiple Drosophila species, suggesting functional importance. The cellular and intracellular localization of these lncRNAs throughout oogenesis will be determined using single-molecule fluorescent in situ hybridization (smFISH). Second, oogenic phenotypes will be characterized for lncRNA mutants. Standard mutational approaches like targeted gene deletion are often insufficient for investigating lncRNA function, as overlapping DNA regulatory elements are common and can confound interpretations. Instead, this proposal aims to develop and implement mutational approaches that specifically target the lncRNA transcript. Third, the mechanisms of lncRNA function will be characterized. Comparative RNA-Seq experiments between lncRNA mutants and wild-type flies will reveal specific regulatory targets of lncRNAs and whether the lncRNAs function in cis or in trans. RNA/DNA double-FISH will be used to confirm regulatory interactions of lncRNAs and their targets, and treatment with an RNA polymerase II inhibitor followed with smFISH will reveal whether lncRNA activity is due to the RNA product or nascent RNA transcription itself. The successful completion of this proposal will provide significant insights into lncRNA biology. As many lncRNAs are also associated with diseases like cardiovascular disease and cancer, knowledge gained from Drosophila will prove valuable to understanding how lncRNAs contribute to human disease.